Apparatus and associated methods for use in microbiological, serological, immunological, clinical-chemical and similar laboratory work

ABSTRACT

For microbiological and other laboratory work in which an active substance is contacted with a substrate there is provided a container for the substrate and a divided carrying body with respective separated sections in sealed contact within the container such that the sections form separate regions with isolated substrate portions in each of the regions. The active substance is supported by the carrying body and is immersed in the respective isolated substrate regions so that it can be diffused into the substrate portions.

CROSS-RELATED APPLICATION

This application is a continuation-in-part of Ser. No. 731,103 filedOct. 8, 1976 now U.S. Pat. No. 4,140,582 which is in turn a continuationof Ser. No. 602,745 filed Aug. 7, 1975 and issued as U.S. Pat. No.3,985,608 which is in turn a continuation of Ser. No. 414,939 filed Nov.12, 1973 now abandoned which in turn is a division of Ser. No. 33,594filed May 1, 1970 issued as U.S. Pat. No. 3,791,930.

BACKGROUND OF THE INVENTION

This invention relates to a new technique permitting rationalization andautomation of microbiological, serological, immunological,clinical-chemical and similar laboratory work.

Before the invention is explained, it will be expedient to give somegeneral background information.

Bacteria are grown in substrates (media) which are either liquid (broth)or of more or less solid consistency. Growth in liquid substrates takesplace throughout the entire substrate, while in solid substrates thegrowth usually takes place on the surface.

The solid substrates are made from the liquid by adding agar (or someother substance, such as gelatine) in various concentrations, or throughthe coagulation of albumin. There are a number of different types ofliquid and solid substrates. Often they have a common basic composition,but otherwise the composition changes according to which substances arerequired for the growth of the bacteria in question.

Many, though not all, bacteria decompose and utilize matter in differentways. This can be registered by various chemical or physico-chemicalreactions. As an example may be mentioned the fermentation or splittingof various types of so-called "sugar" (hereinafter called sugar) bybacteria. This fermentation, considered for the whole series of sugartypes in question, is often characteristic of the individual type ofbacterium, and frequently differs from one bacterium to the other. Thiscircumstance is widely exploited as an aid in distinguishing bacteriafrom one another.

When a bacterium decomposes a sugar to acid products, the pH value inthe substrate decreases, as may be observed by a change in the color ofan added indicator. If, for example, a sugar is added to a solidsubstrate as a fermentation basis, and bromothymol blue is added as anindicator, the result of inoculation with bacteria that break downsugar, and with bacteria that do not, is as follows; The sugar-splittingbacteria will grow on the surface of the substrate and form yellowcolonies, while the non-splitting bacteria will form colonies of thesame blue color as that of the substrate before inoculation. Yellow andblue colonies may lie close together, side by side, on the surface ofthe substrate.

A corresponding liquid substrate will change its color to yellow ifsugar-splitting bacteria grow in it, and will remain blue if the growthis purely of non-splitting bacteria.

When, as an aid to classification, fermentation determinations arecarried out, liquid substrates are usually employed.

Since one of the objects of the invention is to rationalize fermentationtests-without limiting its scope to this field-a more detaileddescription will be given of how a fermentation test is carried outaccording to a known method.

A pure culture of the micro-organism to be classified by thefermentation test must be prepared. Samples from this pure culture areinoculated into broths which have already been poured into test tubes.These broths contain an indicator and various types of sugar. The testtubes are closed with plugs or caps. The test tubes containing thebroths are placed in racks and incubated for growth and reaction. Theresult is read by inspecting each tube, and is noted by hand.

This method requires that the test tubes be placed in the racks by hand.Each tube of broth contains only one kind of sugar, but the number ofkinds of sugar, and thus of tubes, may often be comparatively high. Thismeans that handling the tubes and segregating them according to type ofsugar when they are to be arranged in rows in the racks, can be arelatively laborious process. Furthermore, there is room for confusionand error.

The inoculation process is also relatively laborious. The inoculation ofeach test tube containing the sugar broth involves removal of thestopper, the upper edge of the tube being sterilized by a flame toremove any possible microbial contamination, and the stopper beingreplaced.

The test tubes, which are often of glass, must, after having been read,go through a time-consuming and laborious process before they can beused anew. First they are put in an autoclave to kill the cultures. Thenthey are emptied, washed, and rinsed, whereupon they are fitted withstoppers. After this they are sterilized and are once again ready toreceive new charges of broths containing various types of sugar andindicator.

The preparation of the substrates to be put into the test tubes alsodemands time and labor. First, the broth must be prepared and then, toportions of this, a number of different types of sugar have to be added.These mixtures are then poured into test tubes and duly sterilized. Thetubes are marked to indicate the type of sugar therein. They are thenplaced in a refrigerator, ready for use.

The test tubes require a good deal of space on account of theirrelatively large size and number. This makes itself felt in thelaboratory work, during storing in the refrigerator and incubator,storing and transport from producer to user. In addition, the racksrequire storing and cleaning.

The fermentation test described above can also be carried out bypositioning prefabricated bodies carrying the various types of sugar ina known solid substrate in a dish or container. The indicator may beconveniently present in the sugar bodies, or, alternatively, it may, inknown manner, have been added to the substrate in advance.

Before the bodies are positioned, the substrate surface in the dish isevenly inoculated all over with a suspension or broth containing a pureculture of the micro-organism.

After the bodies have been introduced onto the surface of the substrate,the sugar--and the indicator if this has been added to the bodies--willdiffuse out into the substate. This takes place relatively fast in thesubstrates normally used. As a result, a successful reaction will notoccur if the sugar is applied via a paper disc into which it has beenabsorbed, or via a tablet into which the sugar has been mixed. Thediffusion must be delayed or limited.

SUMMARY OF THE INVENTION

According to the invention, the substrate around the applied body islimited, so that the sugar diffusing out will remain inside aperipherally closed chamber or confined region and equalize itself therewith an evenly distributed concentration. Thus, this final concentrationis dependent upon the volume of substrate enclosed and upon the quantityof the sugar applied.

A more detailed description according to the invention, will be givenhereafter. The limitation is achieved by means of supporting elementswhich encompass or confine a space closed laterally on all sides. Thesupporting elements are pressed down into the solid or semi-solidsubstrate, preferably so that their lower edges reach the bottom of thedish, while their upper edges preferably protrude somewhat above thesurface of the substrate. In other words, the supporting element shouldpreferably be somewhat higher than the depth of the substrate, which isgenerally about 0.5 cm or less. The diameter can be, for example,0.5-1.5 cm or some other suitable size. The supporting elements may beopen or closed at the top.

The supporting elements contain those types of sugar which are to takepart in the reactions. Suitable concentrations and quantities of thesugars, for example, absorbed in filter paper or mixed into a suitablemass, are placed, during manufacture, in an expedient manner in thesupporting elements. This can be achieved, for example, by means of alining containing the sugar types and fixed to the inside of the wallsof the supporting element. This lining may, if desired, rest on a lowerinner shelf or step just above the lower edge of the supporting element.Alternatively, the sugars can be placed in hollows in the walls of thesupporting element, in which case internal wall lamellae are perforatedso that diffusion can take place into the space actually enclosed by thesupporting element. The bottom of the wall cavity may convenientlyconsist of the solid lower edge portion of the supporting element.

In addition to the walls which surround the supporting element to carrythe types of sugar, internal installations in the supporting element mayalso be used for the same purpose. Thus, in the center of the supportingelement, there may be mounted a torpedo-, projectile- or star-shapedpart, or other similar parts of expedient shape. They can be fixed, forexample, by means of a supporting spider carried by the upper edges ofthe supporting element. Furthermore, the supporting element can havepartition wall sections of various shapes which fully or partlysubdivide the space which the supporting element actually surrounds.These partition walls, and in fact all internal installations, may, likethe surrounding walls, be of hollow construction with perforated walllamellae.

After a supporting element containing a sugar has been placed in thesubstrate, diffusion takes place in the part of the substrate enclosedby the walls of the supporting element. In the case of supportingelements with perforated internal wall lamellae, diffusion takes placethrough these holes.

The amount of sugar which in one or another of the methods mentioned isintroduced together with the supporting elements during the productionprocess, is so adjusted that after diffusion into the substrate borderedby the walls of the supporting element, the sugar will assume aconcentration suitable for the fermentation tests to be carried out.

When carrying out fermentation tests, good results will be achievedirrespective of the geometrical form of the cross-section of thesupporting element, but even though this is so, it may be of interest todiscuss the shape of the supporting element in relation to diffusionequilibrium. The shape of the supporting element and the positioning ofthe sugar affects the rapidity with which the final concentration of thesugar is established in the substrate in the supporting element. Acircular supporting element, with the sugar placed along the wall,surrounds a quantity of substrate which will be relatively slowlypenetrated compared to non-circular shapes due to the fact that a circlehas a relatively large area compared to its circumference. Placing thesugar in the middle of the round supporting element and/or alongpartition walls in addition to along the inner wall of the supportingelement entails a more rapid state of diffusion equilibrium. Also, forexample, a narrow rectangular supporting element, with the sugar placedalong the walls, will be favorable for a relatively rapid establishmentof the final sugar concentration in the substrate enclosed. With certainreactions other than fermentation tests, the choice of an expedientshape of supporting element may have practical consequences for thecourse and outcome of the reactions.

An actual or "positive" fermentation reaction is revealed by change ofindicator color after incubation at 37° C. for a suitable period, whileno or "negative" reaction is characterized by no change of color. Theresults of reactions are easy to read.

Gas production may be detected by development of air bubbles afterinoculation into the depth of the substrate in the case of, for example,glucose as the active substance. In the case of a solid agar substrate,gas production will manifest itself by breaking up the substrate.

When it is desired to maintain anaerotic conditions during testing, theinvention lends itself readily to such conditions by providing a sealinglayer, for example, of paraffin at the top of the substrate within thesupporting element by addition of the paraffin after the supportingelement has been inserted into the substrate to the bottom of thecontainer.

In a container containing a substrate, for example, a Petri dish, anumber of supporting elements are placed at suitable mutual distances.Each supporting element contains one type of sugar, and thus all thesupporting elements together represent the different types of sugar withwhich the fermentation test with the bacterium culture in question is tobe made.

It is convenient to supply the supporting elements to the substrate froma dispenser similar to those normally used for positioning paper discscontaining antibiotices. The supporting elements containing the sugarsare stored under sterile conditions, stacked on top of one another inmagazines, each magazine containing supporting elements having the samekind of sugar. In the dispenser or depositing device there is room forseveral magazines side by side, each magazine containing a differenttype of sugar.

Especially if delicate substances are contained in the supportingelements, it may be preferable to store them not only under sterileconditions in magazines, but also vacuum-packed.

After the supporting elements have been positioned on the surface of thesolid substrate they may, by hand or machine, be pressed so that theypenetrate the substrate and establish contact with the bottom of thedish, with the help of, for example, a plunger or similar device whichpresses down all the supporting elements in one and the same operation.The upper edge of the supporting elements, according to the invention,should preferably protrude somewhat above the surface of the substrate,so there is usually no danger of the surface of a plunger contacting thesubstrate.

If required, the supporting elements may be provided, at their loweredges, with small anchoring feet in order to prevent rebound from thesurface of the substrate.

The supporting elements may be mounted in a supporting skeleton by meansof which they can be simultaneously pressed to penetrate the substrate.

In accordance with the invention in U.S. Pat. No. 3,843,450, thesupporting elements may be made to respond to magnetic forces or, inother words, they may be made magnetically responsive. By magneticforces, they are attracted to penetrate the substrate and to makecontact with the bottom of the dish. If required, the magnetic force maybe applied after the supporting elements, by gravity, have landed on thesurface of the substrate. The magnetizable parts can be placed, forexample, inside the wall of the supporting element as its lower part oredge, and around the entire circumference. Alternatively, for example,the entire wall of the supporting element may be ferromagnetic. Thismaterial may conveniently have the form of an unbroken iron ring orsleeve, or also of granular parts, the maximum size being determined bythe thickness of the tubular wall.

Below and along the upper, outer edge of the supporting elements, theymay be provided with a collar of iron of suitable size and shape inorder to increase the response of the supporting element to the magneticfield. If required, the collar may carry slim, downwardly projectingsupporting studs extending outside the element and terminating flushwith the bottom thereof. Their number may, for example, be four, andthey may expediently be placed opposite one another so as to offer abalanced support.

It may be convenient to make the lower edge of the supporting elementfairly sharp or bevelled so as to facilitate penetration of thesupporting element through the substrate.

The supporting elements are discarded after use. Except for the abovementioned magnetizable components, they can, with advantage, be made ofplastic or the like, which is destroyed by burning.

The sugar fermentation reactions are not critically dependent uponcomplete contact or sealing between the bottom of the dish and the loweredge of the supporting element.

Even a roughly cut lower edge of a supporting element ensures reactionsof just as good quality as those with very good contact. Nevertheless itis possible, if desired, to establish adhesion between the lower edgeand the bottom of the dish.

The identities of the reactions are determined automatically orsemi-automatically in accordance with the invention described in U.S.Pat. No. 3,843,450 by means of defined positions.

Also the outcome of each reaction is a function which may be read bymachine in accordance with U.S. Pat. No. 3,843,450. The readings aremade as indicated in the patent, preferably under visual control. Thedata machine used indicates in a simple manner whether a reaction ispositive or negative, or the degree of positivity shown by thereactions. In such a case as this, with clear, distinct color reactionsand well defined criteria for determining the outcome of a reaction, itmay, in accordance with the above mentioned patent, be possible to takethe readings fully automatically.

Conventionally, when carrying out fermentation tests, one investigationis made for each type of sugar. A defined, convenient concentration ofthe sugar is used. Investigations carried out by the inventor indicatethat it would be possible to achieve a better differentiated diagnosticexpression if several concentrations of the particular sugar were usedin the test. Such a method would be prohibitive with the working methodsavailable by present day techniques. Even when only one concentration ofeach kind of sugar is used, fermentation tests impose a heavy load onthe laboratories.

The invention, however, makes it possible in a simple and labor-savingway to carry out a fermentation test with several concentrations of onekind of sugar. Apart from using a separate supporting element for eachsugar concentration, it is possible to use one supporting element forall concentrations, the supporting element being divided into separatechambers to carry the sugar concentrations. The supporting element mayin such a case, for example, have the shape of a relatively longrectangular structure divided into consecutive transverse rectangularsections. Each section contains a given concentration of the sugar insuch a way that there is a successive increase or decrease in the sugarconcentration from section to section in the long rectangular supportingelement. This supporting element represents therefore a particular typeof sugar, and its sections, various concentrations of that sugar.

The type of sugar is identified by the position of the rectangularsupporting element as such, and the concentration of sugar where apossible fermentation first occurs is determined by the position of thechamber concerned in the section row.

The supporting element under discussion may also be used with differenttypes of sugar in the chambers.

However, as mentioned above, the invention is not restricted to use infermentation tests, but is applicable to a large number ofmicrobiological, serological, immunological, clinical-chemical andsimilar tests, where the reactivities of the various active substancesand agents applied to the substrate, i.e., changes in growth and/orother reactions between the active substances and agents, are thesubject of the investigations.

For example, the invention could be applicable to sensitivitydeterminations of bacteria towards antibiotics (and chemotherapeutics).

Today such determinations are usually made by means of discs containingantibiotics. A mechanical and automatic embodiment of this method isdescribed in U.S. Pat. No. 2,843,450. The principle of the antibioticdisc method is to measure the diameter of the inhibition zone around theantibiotic disc and use this measurement as an expression of thesensitivity of the bacterium towards the antibiotic concerned, of whichthe disc contains a given amount.

In contrast to this, the present invention permits sensitivitydeterminations to be carried out in a simple manner by means of atitration technique. In this manner, a quantitative determination isachieved in terms of different concentrations of an antibiotic. In somecases this is more advantageous than the conventional method involvingmeasuring the size of the zone diameter. The methods supplement oneanother.

According to the invention, supporting elements containing variousconcentrations of any antibiotic may be manufactured in advance for usein connection with the quantitative technique discussed above. In atitration test a given series of supporting elements is used for eachantibiotic in such a way that the concentration of the antibiotic inquestion increases from one supporting element to the next. In additionto individual supporting elements, a supporting element subdivided intosection chambers, as described above, can also be used for this purpose.The chambers have been supplied in advance with various concentrationsof an antibiotic increasing in steps from chamber to chamber.Observations are made of the lowest concentration which inhibits growth,assuming that the bacterium being investigated is sensitive towards theantibiotic in question.

Today, if it is desired to carry out a quantitative sensitivityexamination of the kind mentioned, this must be done in a series of testtubes containing broth, or in containers holding solid substrate,containing concentrations of antibiotic increasing in steps. On accountof the labor this involves, this present-day technique would beprohibitive as a general routine method. Yet, in spite of the amount ofwork involved, it is used when determining the sensitivity of tuberclebacilli. This is due to special circumstances to be described below. Theinvention, however, makes it possible, in a simple manner, to make useof a titration technique for determining the sensitivity of tuberclebacilli.

The reason why sensitivity determinations of tubercle bacilli towardsantibiotics differ from the sensitivity determinations of otherbacteria, is the relatively very slow growth rate of tubercle bacilli.One consequence of this fact is that it is not convenient to determinethe sensitivity of tubercle bacilli by the antibiotic disc method in theconventional manner.

However, the present invention makes it possible to determine thesensitivity of tubercle bacilli also by measuring the size of the zoneinhibited. This can be achieved by using a relatively long and narrowrectangular supporting element as a support for an antibiotic. Theantibiotic is fixed to the inside of one of the short sides of thesupporting element. The diffusion of the antibiotic concerned is guidedby the shape of the supporting element in a compressed volume along thelongitudinal axis of the rectangular supporting element. The length ofthe zone inhibited is a measure of the sensitivity. The supportingelements may be open or closed at the top, the closure, when present,being in the form of a flat transparent roof. This cover will delay thedesiccation of the substrate and reduce the risk of contamination duringthe incubation period of several weeks at 37° C. which is necessary forpermitting the growth of the tubercle bacilli to develop. In itself, thesealing of the substrate dish is not entirely sufficient to affordprotection against a certain degree of desiccation during such a longperiod of incubation. Additional sealing of the dishes is required (forexample with tape) or they can be incubated inside plastic bags orboxes. These measures can be rendered unnecessary by using supportingelements closed at the top as mentioned above.

This invention also lends itself to an expedient application of aserological technique, particularly an immunodiffusion or precipitationtechnique. This very important technique has a multifarious andincreasing application, but it is laborous and difficult to use inseries tests. With the help of this invention, this technique will bemade available in a great many fields for routine laboratory work andwill, to a significant degree, contribute to increasing the breadth ofinformation concerning laboratory investigation and research and toenhance their significance.

Instead of inoculating the substrate (preferably at the surface) with anagent consisting of a bacterium culture, it is possible, in this case,to spread a quantity of an agent consisting of antigen or antibody overthe surface of the substrate, or mix it in with the substrate beforepouring it into the dishes, all according to whether the supportingelements contain antibody or antigen respectively. When employing aquantitative precipitation technique, the prefabricated supportingelements contain graded quantities of the said substances.

It is only necessary for the substrate to be a nutrient when microbialgrowth is to take place. In addition to the compositions referred toearlier, it may be made on the basis of cellulose, starch or othersuitable substances. A polyacrylamide gel may also be used. It is atransparent and inert gel, and it is possible to vary the concentrationof acrylamide over a wide range in order to have gels of differentporosity. Thus, larger pores are provided by lowering saidconcentration. The gel, as well as other gels, can in some instanceswith advantage be mixed with other gels, such as an agarose gel. Themost common gel is an agar gel which possesses substantial strength evenwith very high water content. Agar is inert chemically and is suitablefor examining diffusion tests. Agarose is a purer product than agar andgives more transparent gels. It has even a higher gel strength thanagar. Lowering the concentration of agar and agarose and other gellingcomponents as well, gives gels of larger porosity. In case ofmicrobiological testing the substrate will contain a nutrient, althoughnot necessarily so if the articles applied to the substrate serve asvehicles for the nutrient, such as growth requirements and growthpromoting factors. Prior to inoculation, the substrate inmicrobiological testing should be sterile, although not necessarilystrictly so if a relatively heavy inoculum is used in a relatively shortincubation period.

Generally speaking the composition of the substrate shall consist of amaterial which permits diffusion and gives it a solid or semi-solidconsistency. The latter is usually somewhat elastic, but it may also beplastic. Furthermore, the substrate must be penetrable by the supportingelements.

It has been reported by Hitchens in J. Inf. Dis. 29, 390, 1921 and byFalk et al. in J. Bact. 37, 121, 1939 that in the case of a number ofdifferent bacteria, anaerobes as well as aerobes, bacterial growth couldbe readily detected in semi-solid agar substrates. In fact, a more rapidand luxuriant growth was observed by using such semi-solid agarsubstrates than by using plain broths or solid agar substrates.

The substrate may be stratified, for example, having a solid agar as abottom layer and a semi-solid agar as a top layer. In such a substratethe supporting elements will be kept in position by the side supportprovided by penetration into the solid agar, whereas the semi-solid agarwill permit rapid diffusion and equilibrium of the active substances,and also more rapid and luxuriant growth.

In some cases, it may be sufficient to use supporting elements that arepartially open laterally, for example, rectangular supporting elementsfrom which the one short wall is missing, or which form open curves.Supporting elements of such a shape will constitute something betweenthe fully open ones, known per se, for example, paper discs, and thosedescribed above which surround a space closed on all sides. The materialwhich is to diffuse is fixed to the far wall of the supporting element,and the diffusion, in its first phase, is guided in a fixed direction bythe walls of the supporting element. When the diffusion has extendedbeyond the bounds of the supporting element, it will spread in alldirections.

The supporting elements may be of various shapes, each suited forspecific diffusion experiments. This may apply to variousimmunodiffusion tests but is not limited to this particular application.Thus, it may be very useful to consider and make use of the sameexpedient when bacterial growth is involved in the experiments, forexample, when testing synergism or antagonism between antibioticstowards bacteria. This investigation is an important supplement to theusual sensitivity determinations, and the possibility now available toinclude such tests in normal routine investigations is a large andimportant step forward.

The invention may also be used in connection with investigationsinvolving growth factors. In advance, growth-promoting substances can beplaced in the peripherally, for example, fully closed supportingelements. After placing the supporting elements in the inoculatedsubstrate there will, through diffusion of the substances mentioned intothe basic substrate in the dish, be produced new substrates in thevarious supporting elements, different from that originally present inthem. These new substrates consist of the basic substrate and one ormore additional different components all dependent on the number ofgrowth-promoting substances placed in the supporting elements. The basicsubstrate in the container may be water, whereas dehydrated selective ordifferential substrates may be located or placed along or in the wallsand/or internal installations of the supporting elements. Differentsubstrates are thus produced in one dish by reconstitution. Observationsare then made of the inoculated bacterium culture in the differentsupporting element to see whether they react with abundant, unchanged orpossibly reduced growth, in response to the addition of the varioussubstances. The technique may be used in connection with diagnosis orfor purely experimental purposes.

The inhibitory effect of dyes and other inhibitory chemicals on bacteriaare known from various studies. The invention makes it possible in arapid and automatic way to use such dyes besides antibiotics and growthpromoting substances making complex combinations of nutrients andchemicals to study the growth pattern or profile determination ofbacteria to provide bacterial identification. Enzymatic profiles mayalso be used in such identification. Since the invention makes itpossible to establish a primary noting by machine of bacteriological orother data in machine or on-line contact with a central data processingunit, the invention permits automatic computer assistance in handlingthe data concerning the wide range of biochemicals which may be involvedin the "metabolic fingerprinting" and enzymatic profile identificationof bacteria.

Reactions may be detected more readily and rapidly by applying adistinction promoting substance, such as a tetrazolium salt or dye tothe reaction areas of the substrate. Thus, a tetrazolium indicator maybe applied to the substrate prior to the outset of a test or during thefirst steps thereof which test may be, for example, determination ofbacterial sensitivity to various antibiotics, or preferably saidindicator may be applied after 6-7 hours incubation period permittingthe results to be read shortly after (V. J. Boyle et al., AntimicrobialAgents and Chemotherapy, 3,418, 1973). The color development induced bythe reduction of the indicator where growth occurs on the substrate, isreadily detectable and causes delineation of distinct zones ofinhibition even when growth is slight after the shortened period ofincubation indicated above.

Some test methods require a secondary application, to the reactionregion, of a particular substance (or substances) after a suitableperiod (or periods) in the course of the reaction, for example, afterincubation for growth has taken place. Such a technique may be carriedout by means of the invention. By using a dispenser, the secondaryapplication can be made in the same pattern as the one used for thefirst positioning. For example, paper discs or other suitable bodiescontaining the substance or substances to be added, may be placed in thesupporting elements. Another way of making secondary applications is touse a device having a number of inoculation points or pipettes arrangedin the same pattern as that in which the supporting elements wereplaced.

This technique, characterized by the secondary application of substancesafter suitable periods of time have elapsed, may also be useful in thecase of a quantitative method utilizing supporting elements whichcontain a substance present in a graded series of concentrations fromone supporting element to the next. The possibility is thereby opened ofadapting the method to different, relatively complicated titrationtechniques which today are carried out with liquid substances in testtubes. This also applies, for example, to reactions such as those inwhich red blood corpuscles are used to bring about haemolysis orhaemagglutination as an aid to determining the titration end-points.Secondarily, the red corpuscles or other particulate material such ascoated or sensitized latex particles or bacteria may be placed on thesurface of the substrate within the supporting elements, where they willshow very rapid and distinct agglutination in the case of serologicaltesting, or they may have been admixed with the substrate in advance.

In some cases, the lining of the carrying body (or of the innerinstallation) may be bare i.e. lack any substance in which case the"secondary" application will in fact be the first contact of the activesubstance with the agent. The substrate may have a very simplecomposition. It may be adapted to existing requirements. Theconcentration of, for example, the agar may be made to suit prevailingconditions. It may, for example, be relatively low. The substrate mayalso contain substances other than the red corpuscles mentioned above,which take part in the reactions in constant quantities from reaction toreaction.

Alternatively, titration techniques may be employed by using the agentssecondarily applied in graded concentrations, while the substancescontained in the supporting elements in this case form a series with aconstant concentration level.

Each supporting element need not contain only one substance, but maycontain several. These substances may be pooled, or positionedseparately at different and isolated places in the supporting element,in order to prevent a reaction from taking place between the substancesbefore diffusion into the substrate.

The supporting elements may also be produced without substances beingadded during the manufacturing process, but with the possibility ofadding required materials after the supporting elements have been placedin position in the substrate. With this object in view, the supportingelements may, for example, be supplied with a funnel-shaped inputchannel leading to a cavity in the wall of the supporting element where,in advance, a piece of absorbent paper may have been placed. Thesesupporting elements too, may be of various shapes, each of a designsuited for various specific diffusion experiments, for example, forexamining precipitation patterns, testing synergism or antagonismbetween antibiotics, or when examining growth factors. They may enclosea hollow space peripherally closed on all sides, or they may be partlyopen. The purpose is to give research workers a technical aid that canimprove possibilities and facilitate their work in the wide field ofexperiment covered by diffusion techniques and microbiology.

Furthermore, the invention makes it easy to determine the level ofconcentration of antibiotics in tissue liquids and to carry out otherassays.

The invention may be used in a quantitative and automatic techniquebased on spectrophotometric readings through the contents within thepositioned supporting elements. This may apply, for example, toclinical-chemical tests. The system may be used for carrying outautoanalyzer work. Depending on how many supporting elements arepositioned by the dispenser at a time, the invention may easily be usedin connection with a 12-channel run or more. The invention may, forexample, be used for screening tests.

The system of the invention is very flexible in use. It may be used bothwith substances which disperse rapidly, and with those which diffuseslowly in the substrate selected. This opens great possibilities formany different reactions and types of reaction to be adapted to thetechnique thus providing the inherent advantages which will be discussedin more detail below.

The system of the invention utilizes principles which were defined andexplained in U.S. Pat. No. 3,843,450. The advantages discussed in thesaid patent will also benefit the present invention. They are concernedwith the automation of laboratory work and primary noting of data takinginto consideration a technically desirable, or even necessary human andvisual guidance and control. They are further concerned with thepossibility of establishing direct contact with an electronic dataprocessing unit with the enormous advantages offered thereby. Due to thepresent invention, these principles may find a more general andcomprehensive application and will benefit a wide range of variousmicrobiological, serological, immunological, clinical-chemical andsimilar tests and research in diffusion techniques in biological andchemical fields.

A technique adapted to the invention will benefit by advantages inaddition to those mentioned above. These advantages relate partly to theactual laboratory work itself, partly to the preparation of thesubstrate, to cleaning, partly to questions of storage and transport andfinally to safety aspects in the laboratory work concerned, in that therisks of errors and confusion are greatly reduced.

A more detailed explanation of these conditions will be given withreference to the technique used in the bacterial fermentation of sugars,mentioned above. The invention involves only one inoculation for theentire series of sugars, as opposed to the system in use today, whichrequires one inoculation for each type of sugar, or, in some cases, fortwo or three types. Furthermore, there is only one cover, namely the lidof the Petri dish, to take off and replace, as compared with the presentpractice of using a plug or cap for each broth test tube correspondingto the individual sugar types inoculated. In addition, all thefermentation reactions are collected in one handy, convenient andspace-saving unit, the Petri dish, as compared with the present somewhatuntidy, laborous and cumbersome system with all the individual brothtest tubes to be handled and spread out one after the other in the holesof a rack. A further disadvantage in this connection is that theindividual test tubes containing the various types of sugar are alikeand can only be distinguished by means of a label indicating the type ofsugar contained. Relatively speaking, the number of substrate dishes tobe handled is small, and they are all of the same type, whereas thebroth-containing test tubes are many, all externally the same, yetcontaining various types of sugar.

Whereas the reading and noting by the new technique may be carried outautomatically under visual control or fully automatically according toU.S. Pat. No. 3,843,450, these operations are very laborious andtime-consuming when conventional techniques are used, in which thevarious fermentation tubes, standing in close rows and columns in a rackarranged according to the kind of test and the type of sugar, have to beremoved manually one by one for taking readings of the outcome andobserving the identity, whereafter the data are noted by hand.

With the conventional technique, the risk of error is relatively large.This applies to the reading and noting of data. It also applies to theinoculation. This is repeated for every broth test tube in the series.It is possible to overlook a tube, or it may be inoculated from a loopwhich does not contain sufficient inoculate to promote growth. Thisleads to erroneous results.

Errors and confusion may also occur at an earlier stage. They may occurwhen the technical assistant fetches the test tubes containing the sugarbroths. These are kept in a refrigerator and are grouped separately foreach type of sugar. It may however happen that the test tubes that havenot been used are put back in the refrigerator in a wrong group,inasmuch as the tubes in all groups have a similar outer appearance.Each test tube must therefore be checked.

Furthermore, errors and confusion may arise in the substrate departmentwhen the broths containing the various types of sugar are beingprepared. Mistakes may also be made in affixing labels and when thetubes are transported to the refrigerator.

The fact already mentioned that a relatively large number of brothscontaining different sugars are required for fermentation tests,increases the possibility of error. This applies particularly when thereare several specimens to be examined. In contrast to this, the techniqueaccording to the invention means that only a relatively small number ofPetri dishes have to be handled, and all of the same type.

The invention will result in a considerable saving in space both duringstorage in the laboratory and during internal and external transport anddispatch. The small supporting elements can be housed in magazines ascompact units as compared to the collection of relatively very largeindividual test tubes, which are awkward to handle. These require standswhich take up a lot of space and have to be kept clean and free fromcontamination. Often the tubes are of glass, and must, after use, be putin an autoclave, washed and sterilized before they can be used again.

On the other hand, it is very simple, after use, to remove and destroythe Petri dishes and with them the supporting elements placed in thesubstrate. When removing them, it is of considerable advantage to beable to handle the small units, requiring little space, which theinvention makes possible.

It is also very advantageous that the invention makes it possible to usea very simple and standardized substrate for various tests andexperiments.

Although other modifications or versions of the fermentation test exist,none of them--or other laboratory methods currently in use orknown--have any relation to the principles or system of the invention.

In the following, the invention will be described in more detail withreference to the drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a Petri dish containing substrate, seen from above and insection along the line I--I in FIG. 2, and provided with supportingelements according to the invention, for carrying out the experimentsdesired,

FIG. 2 is a view in cross-section along line II--II in FIG. 1,

FIG. 3 is a vertical sectional view through one embodiment of asupporting element according to the invention,

FIG. 4 is a vertical sectional view through another embodiment of thesupporting element,

FIG. 5 is a vertical sectional view through another embodiment of thesupporting element,

FIG. 6 is a side elevational view of another embodiment of thesupporting element,

FIG. 7 is a top perspective view of another embodiment of the supportingelement,

FIG. 8 is a side elevational view, partly broken away and in section, ofanother embodiment of the supporting element,

FIG. 9 is a vertical sectional view through another embodiment of thesupporting element,

FIG. 10 is a top perspective view of another embodiment of thesupporting element,

FIG. 11 is a top perspective view of another embodiment of thesupporting element,

FIG. 12 is a section taken on line XII--XII in FIG. 10,

FIG. 13 is a top plan view of another embodiment of the supportingelement,

FIG. 14 is a side elevational view of the embodiment of FIG. 13,

FIG. 15 is a top perspective view of another embodiment of thesupporting element,

FIG. 16 is a perspective view of a divided element and container priorto their engagement,

FIG. 17 is a longitudinal section of the divided element and containersubsequent to their engagement,

FIG. 18 is a perspective view of a modified arrangement of the dividedelement and container prior to their engagement,

FIG. 19 is a sectional view of a portion of the divided element andcontainer in FIG. 18,

FIG. 20 shows the divided element and container of FIG. 19 in engagedrelation,

FIG. 21 is a diagrammatic transverse sectional view showing apparatusfor photometric analysis of the assembly shown in FIG. 20,

FIG. 22 shows another embodiment similar to FIG. 18 of a modifieddivided element and container prior to their engagement,

FIG. 23 is a sectional view of the divided element and container in FIG.22 in engaged relation,

FIG. 24 is an enlarged view of a portion of the element and container ofFIG. 22 prior to engagement showing a modified sealing means therefor,

FIG. 25 shows the engaged relation of the divided element and containerin FIG. 24,

FIG. 26 shows a further modified arrangement of the sealing means priorto engagement of the elements,

FIG. 27 shows the assembly in FIG. 26 after engagement of the elements,

FIG. 28 shows an arrangement similar to FIG. 26 with a still furthermodified sealing means,

FIG. 29 shows the elements of FIG. 28 after assembly,

FIG. 30 shows another modified arrangement of the sealing means,

FIG. 31 shows the arrangement of FIG. 30 after assembly of the elements,

FIG. 32 is a sectional view showing a carrying element with sealingmeans therefor in a position prior to engagement of the carrying elementwith the bottom of the container,

FIG. 33 shows the carrying element in sealed engagement with the bottomof the container,

FIG. 34 is a sectional view of another embodiment of the carryingelement engaged with the container,

FIG. 35 is a perspective view, partially broken away, of a modifiedembodiment of the carrying element,

FIG. 36 is a longitudinal sectional view through a portion of thedivided element of an assembly including FIG. 35,

FIG. 37 shows a modified arrangement of the assembly as shown in FIG.36,

FIG. 38 is a perspective view showing a further modified divided elementin separated relation with a container therefor, and

FIG. 39 is a sectional view of a dispenser in combination with anassembled divided body and container.

DETAILED DESCRIPTION

The supporting element according to the invention may be used for manytests and experiments, but in the following description there isprimarily given an account of the determination of bacteria on the basisof their fermenting effect on various types of sugar.

The different sugars are added to a solid or semi-solid substrate, whichis inoculated with the bacterium agent to be examined. The sugars arebrought into contact with the substrate by means of a supporting elementor carrying body according to the invention, for example, of the formshown in FIG. 3. The supporting element 3 in this embodiment is atubular carrying body which has on the inside of the wall thereof alining 4 containing a sugar (and an indicator if this is not containedin the substrate) to be used in the experiment. A ferromagnetic ring 5is mounted at the bottom of the tubular supporting element. By means ofone or more permanent-magnets or electromagnets, it is possible to drawthe tubular supporting elements into position through the substrate in aPetri dish. This positioning is shown in FIGS. 1 and 2. Here, the Petridish is indicated by numeral 1. The substrate which has been inoculatedwith the bacterium culture to be determined, is indicated at 2. Sixtubular supporting elements of the type shown in FIG. 3 have been placedin the substrate. Each supporting element 3 has a lining 4 carrying itsown type of sugar. This varies from one supporting element to the nextin the substrate dish. The outcome of the fermentation reaction (whichsugars, if any, are fermenting) is a diagnostic aid when determining thebacterium. Fermentation is revealed by a change in the indicator color.Such a change has been indicated in the substrate which is encompassedor confined by the supporting element designated at numeral 3' inFIG. 1. The fermentation causing the indicator to change color, isinitiated after the various sugars and the indicator, if added to thebodies in the linings 4 of the six supporting elements have difused intothe substrate enclosed by each individual supporting element, thediffusion of the sugar into the confined area producing an equilibriumconcentration thereof in the confined area.

The lining 4 may, for example, be of paper or cellulose, and it rests,in this embodiment, on a step or shelf formed at the lower end of thetubular supporting element 3 by the ring 5.

Instead of the lining, there may be provided a perforated wall lamellawhich forms the inner boundary of a cavity 4' in the wall of the tube 3aas shown in FIG. 4. The sugar is placed in the cavity. It is placedthere either in its original form or it is absorbed in a lining orcoating layer or mixed with an inert mass. It diffused through theperforated lamella and into the substrate encompassed by the tubularsupporting element when this is pressed into the inoculated substrate.

The tubular supporing elements may consist of various suitablematerials, for example, a plastic. The material may containferromagnetic material, either inserted or cast in position, or thesupporting elements may be made of a ferromagnetic material, such assteel.

If, in special cases, more powerful magnetic forces are required thanthose obtainable by the ferromagnetic ring 5 in FIG. 3, (even whenadding additional ferromagnetic material into the wall of the tube) thetubular supporting element may, as indicated at 3b in FIG. 5, have aferromagnetic collar 12 or other suitably shaped member disposed aroundthe upper end of the tube. In FIG. 5 the entire wall of the tubularsupporting element, including the collar, consists of a ferromagneticmaterial. The supporting element has, in this case, a somewhat taperedshape as compared to the one shown in FIG. 3. In FIG. 6 theferromagnetic collar 12' is of triangular cross-section disposed on atubular supporting element 3c of the type shown, for example, at 3 inFIG. 3. Should it be necessary to make the supporting element morestable when it has a ferromagnetic collar at the upper end, thesupporting element may be provided with four supporting legs 13 as shownin FIG. 7.

The embodiment in FIG. 8 has a projectile-like body 10 placed centrallyin the tubular supporting element 3. This projectile-like body issupported from element 3 by a spider 11. The body 10 may be provided, onits outer face, with a coating containing the active, diffusiblesubstance corresponding to that in the lining 4. In this manner,diffusion equilibrium in the substrate mass is rapidly achieved.

Alternatively, different diffusible substances may be used in lining 4and on the surface of the body 10.

The body 10 may be hollow and perforated. The diffusible material maythen be placed in the body either during the manufacturing process or ata later stage. The supporting elements intended for the latter purposeare manufactured without active materials in the bodies 10. These meet ademand in certain situations, for example, in certain experiments wherethe experimenter wishes himself to add the materials to be tested. Thisis achieved by allowing them to drip down into the hollow bodies.

The supporting element as shown in FIG. 9 is suitable for experimentalpurposes connected with the same requirements for use as indicatedabove. The diffusible, active substance is allowed to drip down fromabove, into space or cavity 8, at the funnelshaped, widened part of thetubular supporting element 3. The substances diffuse into the substratemass through a perforated inner part 7 of the supporting element, whilea closed, upper part 9 prevents the substances from leaking out onto thesurface of the substrate. In this way, the substances diffuse into thesubstrate along the wall of inner part 7.

The term "tubular supporting elements" here also refers to supportingelements which are not of circular cylindrical shape, and for example,the supporting elements may be in the form of an elongated box, as shownat 13 in FIG. 10. Such a shape is suitable if it is required to guidethe diffusion in a particular direction. This is achieved by placing theprepared lining 4 on one of the short walls as indicated in the figure.The diffusion will then spread along the body 13 of the supportingelement towards the opposite short wall. If the linings are placed alongone long side or along both sides as indicated at 14, diffusionequilibrium will be rapidly established. As shown in FIG. 12, therectangular supporting element in FIG. 10 has neither base nor cover,and the supporting element may carry a ferromagnetic material as shownat ring 15. Further, the supporting element may be provided with a roofor cover, if desired.

FIG. 11 shows a different embodiment of a supporting element 13a in theform of an elongated, rectangular structure, in which the interior spaceis divided by walls 20 into four equal spaces, each of which may have alining containing a different substance. A section through FIG. 11 wouldhave the same appearance as the one through FIG. 10 and shown in FIG.12.

In the embodiments shown, one of the objects of the invention is to keepthe reaction confined to a certain area. According to the invention, thesupporting elements, may, however, also be so designed that only thefirst stage of the diffusion is controlled and guided in a particulardirection, while the final stage is free and unrestricted. An example ofthis is shown in FIGS. 13 and 14. In this case, the supporting element23 is provided on its outer surface with longitudinal concavities 16having small holes 18. These lead into longitudinal passages 19. Thesepassages may either be furnished with active substances duringmanufacture, or the substances to be used or investigated may be addedduring the experiment. This can be achieved by depositing drops of thesubstances into the longitudinal passages 19.

The substances will, in time, diffuse out into the substrate through theholes 18. As the diffusion spreads further outwards, the direction willbe determined by the shape and size of the curved part. It is not untila later stage, when the dispersal has proceeded beyond the confiningborders, that it becomes uncontrolled in all directions.

As explained with reference to FIGS. 4 and 9, the supporting element mayalso be made with a perforated wall lamella confining a chamber intowhich the substances of interest may be introduced. Alternatively, thesupporting element may be without chambers and perforations in thewalls. In such a case, the substances in question may be added in theform of, for example, a paste in the longitudinal concavities 16, orthey may be contained in a lining or coating layer which, in a suitablemanner, is fixed to the outside of the supporting element.

The supporting element shown in FIG. 13 is hollow in the center. Thesupporting element may also be made without this cavity, but if so, itshould be thin enough to ensure that it does not cause cracks to form inthe surrounding substrate during or after its introduction.

In FIG. 15, a star-shaped supporting element is shown wherein aplurality of flat walls 19 intersect along a common line. Thissupporting element also causes the substances to be diffused accordingto a fixed pattern. The substances in question are placed--or havealready been placed--in the angles 17 formed between adjacent walls 19.

In FIG. 16 there is seen a supporting element 13a of the samecontruction as shown in FIG. 11 positioned above a container 21 ofconforming shape to that of element 13a. The element 21 contains asubstrate 22 (FIG. 17) which may be liquid, semi-solid, or solid. Theelement 13a is insertable into the container 21 and into the substrate22 therein as shown in FIG. 17. The lower edge of the element 13a formsa sealed relation with the bottom of the container 21 so that separatesections 23 of element 13a will form respective separate regions withisolated substrate portions in each of the regions when the element 13ais inserted into container 21 to the bottom thereof. A cover 27 can beplaced on the assembly of the element 13a inserted into container 21 inorder to cover the respective isolated sections.

FIG. 18 shows a somewhat similar arrangement wherein divided element113a is insertable into container 121 as shown in FIG. 20 to formseparate regions 123 corresponding to the number of divided sectionswithin the divided element 113a.

The container 121 comprises upstanding walls 131 which form a shelf 132against which the lower edge 133 of element 113a comes to rest when theelement 113a is inserted into the container 121.

In order to guide the entry of the divided element 113a into thecontainer 121, the element 113a is provided with two rectilinear guides134 at one end and a single rectilinear guide 134 at the opposite end.The guides 134 are connected by webs 135 at the upper edge of theelement 113a. The container 121 is provided with corresponding bores 136in the opposite end walls therein to receive respective guide elements134. The guides 134 and bores 136 are illustrated as being circular butobviously they can be of any suitable cross-sectional shape.

Beneath shelf 132, the container 121 is provided with a number ofdivided sections or compartments equal in number to the divided sectionsin body 113a. These divided sections or compartments come into registrywith the divided sections of element 113a to form separate regions 123with isolated substrate portions in each of the regions. The substrateis present in the container to the level as shown at 137. When thedivided container 113a is inserted into the container 121, it penetratesinto the substrate so that the lining of active material 114 which is onthe walls of the divided element 113a become immersed into the substrateto enable diffusion to take place within the isolated sustrate portionsin each of the regions.

The substrate can be liquid, semi-solid or solid. The substrate can beadded to the container 121 immediately prior to the introduction of theelement 113a therein or it can be previously provided in the container121 and stored therein long before the introduction of the dividedelement. The agent can be inoculated or spread onto the surface of thesubstrate or throughout its entire content. The agent, such as coloniesof bacterial cultures can be emulsified in a liquid substrate, such aswater. The substrate containing the agent can also be body fluid such asserum or urine. Indeed, in some cases the substrate may, in fact, revealwith the subsequent examination with the active substance, that no agentis present.

The container 121 can be supported on a base 140 which is provided withleveling feet 141 at the four corners thereof to ensure a perfectlylevel condition for the substrate. A suitable means 142 is employed tohold the container 121 on the base and as shown in FIGS. 18 and 19, theholding means 142 comprises blocks 143 capable of engaging end walls ofcontainer 121 in clamped relation. Each block is traversed by a bolt 144which rides in a slot 145 provided at a respective end of base 140. Inoperation, one end of the container 121 is brought to bear against asecured block 143 and the block at the other end is thereafter broughtinto clamping contact with the other end wall of the container 121whereafter the bolt 144 is tightened to securely hold the containerbetween the blocks. Alternatively, the clamping means can be in the formof a snap-lock or a spring latch or any other suitable quick releasemeans.

The walls 131 of the container 121 terminate at edges 150 along thelongitudinal sides so as to leave the depending chambers exposed as seenin FIG. 18. The chambers are formed of a transparent or translucentmaterial in order to enable photometric analysis of the results whichtake place within the chambers.

The system for undertaking photometric analysis will be best seen froman examination of FIG. 21 wherein a conventional photometric analyzer isillustrated. Herein, light from a source 201 is directed into a chamberin the container 121 and due to the presence, for example, of asuspension therein due to reaction between the agent in the substrateand the active substance diffused from the walls of element 113a, thebeam of light from the light source is scattered and its measurement canbe taken as a measurement of the reaction e.g. the bacterial populationin the case of a microbiological examination. The scattered light ismeasured at an angle with respect to the original direction of the lightbeam by a photosensor 202. A collimator 203 can be interposed betweenthe container 121 and the photosensor 202. It is to be understood thatall of the chambers can be examined by respective photometric analyzerssimultaneously, or by a single photosensor through the medium of arelative movement between said photosensor and the divided container. Ifthe reaction takes place at the surface of the substrate, the lightsource is directed through the container 121 to the surface of thesubstrate and the photosensor and collimator are positioned to receivescattered light. Other optical examination systems are known in the artand can be employed instead of the light scatter technique as describedabove. By way of example, spectrophotometric, optical densitiymeasurement apparatus or other turbiodometric or nephelometric orradiation detection means can be used.

To one of the chambers in the divided container, no active substanceshould be added, for example, no antibiotic, since this chamber shallserve as a control chamber or reference or blank in the photometricmeasurement. The use of a reference chamber is applicable to allembodiments when a plurality of separate sections with isolatedsubstrate portions is formed. Thereby, the degree of reaction of theagent with active substances can be referred to a common basis, i.e.,the reference chamber where no reaction takes place.

Since it is common in microbiological testing procedures to shake thecontainer to ensure proper suspension of the agent and rapid elution ofthe active substance, any suitable means can be employed for agitatingthe container 121 after insertion of the divided element 113a into saidcontainer.

It is also well known to incubate the substrate to promote thereactivity of the agent and active substance(s), i.e., changes in growthand/or other reactions between the agent and active substance(s), inmany testing procedures and for this purpose the container 121 and theinserted elements 113a can be placed into an incubator prior tophotometric analysis. The incubator may include an agitator means toeffect agitation of the assembly in the course of incubation.

The level of the substrate which has been shown at 137 can be visuallydetermined by the provision of level indicator markings engraved in eachend wall of the container 121. The indicator markings may be arranged inopposite pairs, each pair being optionally given a different color. Themarkings not only serve to indicate the quantity of liquid contained inthe container 121 but also to aid in leveling the container for uniformdistribution of the substrate in each of the chambers in the containerand in the corresponding sections of the divided element 113a insertedinto the container.

With the above construction there is only a minimal risk ofcross-contamination of active substances, eluted from the separatesections of the divided elements, among the isolated liquid substrateportions in the separate regions when the divided element is insertedinto the container. In this respect, the insertion of the dividedelement into the container will be quickly completed which will leavetoo short a time for any cross-contamination. Nevertheless, in order toreduce even further, any risk of such cross-contamination, the level ofthe upper surface of the liquid substrate may be kept just above theshelf at the top of the divided sections or compartments in the lowerpart of the container.

In order to physically safeguard against any possibility ofcross-contamination, the construction as shown in FIGS. 22 and 23 isemployed. Therein the lining 214 containing active substance is placedhorizontally around the inside walls of each divided section 223 of thedivided element 213a at a distance above the lower edge 224 of eachsection, so that the lower edge of the strip 214 will just avoid contactwith the substrate surface 237 when the divided element 213a and thecontainer 221 are joined. If desirable the lining may extend to the topof section 223. In stead of being placed horizontally, a strip may beplaced vertically or in any suitable position on the inside walls ofsection 223. Furthermore, the lining may take any suitable form.

An appropriate agitation will then cause the liquid or semi-solidsubstrate to wash over the strip or lining and elute the activesubstance therefrom.

In order to provide a fluid-tight engagement of the element 213a withthe container 221, an engagement means 240 (FIG. 23) is provided. Theengagement means 240 comprises a continuous bead 241 which extends alongthe lower edge of the element 213a and which fits into a correspondingslot 242 extending along shelf 232 on which the element 213a rests wheninserted within the container. As seen in FIG. 23, the bead is ofcircular section and is deformable in order to be snapped into the slot242 for tight engagement therewith. As a result of the sealed engagementof element 213a with container 221, the lining strip 214 in each section223 will be confined to act with the substrate associated with therespective chamber 225.

FIGS. 24 and 25 show a modified form of engagement means particularlyadapted to sealably engage the lower edge of the divided element withthe container. In the embodiment in FIGS. 24 and 25, the bead 341 isshaped as a portion of a circle in cross-section with a sharp tip 342.The bead is connected by a neck portion 343 to the lower edge of thedivided element. The bead is engagable in the slot 344 in the containerby a snap effect, the tip 342 on the bead fitting into a V-shaped groove345 at the bottom of slot 344. The groove 345 is filled with adhesive.The slot 344 is covered with a foil 346 or other liquid-tight cover.Upon assembly of the divided element and the container, the tip 342 ofthe bead will pierce the foil 346 and the bead will penetrate into theslot so that the adhesive in groove 345 will bond the lower portion ofthe bead 341 in the slot 344 to provide a liquid-tight adhesive sealingin addition to the mechanical snap-lock effect. The ruptured foil 346will also act to prevent seepage of the substrate into the slot duringthe assembly operation.

FIGS. 26 and 27 show another embodiment of a sealing arrangement betweenthe divided element 213a and the container 221 wherein the bead is inthe shape of a rectangular projection 441 which fits into acorresponding rectangular slot 442 in the container 221. The walls ofthe slot are covered by a layer of adhesive material 443 to effectivelybond the projection to the walls of the slot after assembly of thedivided element and the container. A liquid-tight foil 444 is providedat the top of the slot.

FIGS. 28 and 29 show a further embodiment of a sealing arrangementwherein a gasket or sealing strip 541 extends along the lower edges ofthe walls of the element 213a. As seen in FIG. 28, prior to assembly,the sealing strip 541 has an annular cross-section. After assembly, asshown in FIG. 29, the sealing strip 541 has been pressed into sealingengagement with the shelf 232 of container 221 whereby the sealing stripis deformed. The element 213a is forcibly engaged with the container 221by magnetic effect produced by the use of strips 542,543, one of whichis of permanent magnetic material and the other of magneticallyresponsive material, the strips being in proximity with the lower edgeof element 213a and the surface of shelf 232.

FIGS. 30 and 31 show another modified form of the sealing arrangementbetween element 213a and container 221 wherein the bead 641 has anenlarged head which is deformingly inserted into slot 642 in thecontainer 221. The engagement of the bead into the slot is by snap-fitand at the bottom of the slot there is provided a resilient sealingstrip 643 which is deformed by the enlarged head of bead 641 when thebead is inserted into the slot as shown in FIG. 31.

FIGS. 32 and 33 show the provision of a sealing means 31 at the loweredge of an individual supporting element 3 for sealably engaging thesupporting element in liquid-tight manner with the bottom of thecontainer 1. The sealing means 31 is in the form of a suction-cup bead32 of flexible material which, when brought into contact with the bottomof the container forms a sealed connection therewith in the nature of asuction cup connection. In order to promote the liquid-tight sealedrelation between the suction-cup bead 32 and the bottom of thecontainer, an adhesive 33 can be incorporated in a hollow in the suctioncup bead 32. This embodiment is particularly effective when thesubstrate is a liquid or semi-solid and the container has a flat bottom.The bead 32 has an initially tapered, streamlined shape to facilitateits penetration into the substrate and to protect the adhesive 33 in thehollow. The bead 32 is provided with external reinforcements 34extending along the lateral sides partially along the depth of the beadsto give the beads sufficient support to resist the pressure due to thepenetration into the substrate but which are yieldable when the beads 32are urged against the bottom of the container and undergo deformation asshown in FIG. 33.

Although the sealing means 31 has been illustrated in conjunction withelements 3 they can also be effectively employed at the lower edge ofthe elements as shown in FIGS. 10-15.

FIG. 34 shows an arrangement wherein the divided element 313a has eachof the sections 323 thereof constructed in the manner as shown in FIG. 9wherein a perforated inner element or lamella 807 is mounted in spacedrelation from the inner wall of the respective section 323. Eachperforated element 807 rests on a step or shelf 808 formed at the loweredge of the section 323 and defines together with the walls of thechamber a cavity 809 in which active substance is placed. The activesubstance may be directly introduced into the cavity, for example, inthe form of a liquid into a blank lining within the cavity or the activesubstance can already be contained within the lining. The top of theperforated element 807 is closed by a closed upper part 810 whichprevents the substances from leaking out onto the surface of thesubstrate. The depth of the closed upper part 810 will be sufficient topenetrate into the upper surface 137 of the substrate in the container221. Although no sealing arrangement has been shown in FIG. 34, it willbe appreciated that any of the sealing and clamping arrangementspreviously described can be employed herein.

FIG. 35 shows a modified arrangement of the divided element 413a. Theelement 413a is in the form of a divided body as before with theexception that the divided sections are closed at the top wall exceptfor openings 901 which lead into the interior of respective hollow innermembers 902 which are integral with element 413a and which protrudebeyond the lower edge thereof. When the divided element 413a isassembled with the container 221, the hollow inner members 902 projectinto the individual chambers 225 in the container 221 by penetratinginto the substrate in the container 221.

The hollow inner members 902 are closed at the bottom and are providedin the vicinity thereof with openings or perforations 903 so as to allowactive material which is housed in or added to each of the inner membersto diffuse into the confined substrate in each chamber 225.

In order to prevent build-up of pressure in the hollow inner members 902or in chambers 904 at the top of the separate sections in the dividedelement 413a when the divided element is inserted into the substrate inthe container 221, each hollow inner member 902 is provided with a slotor perforations 905 near the upper end thereof to provide communicationbetween the interior of the hollow inner member and the respectivechamber 904 and in the top wall of the divided element 413a there areprovided a number of vent holes 906 providing communication between theambient atmosphere and the chambers 904.

The upper openings 901 of the hollow inner members 902 are aligned andmay be closed by a narrow lid or cover 910 which is slidably engageablein gripped manner with a dovetail slot 911 in the divided body 413a tocover all of the openings 901. A corresponding dovetail slot 911 may beprovided at the opposite end of the element 413a for receiving theremote end of the cover 910.

The vent holes 906 may be covered by a hinged cover or other suitablevalve means for maintaining a closed condition within the dividedsections in the element 413a until such time as a pressure build-up inchambers 904 causes the valve means to open.

FIG. 37 shows the provision of a combined locking and sealingarrangement for the assembly of divided element 413a and container 221.

The combined locking and sealing arrangement comprises an integral cover921 formed at the top of element 513a which includes a depending leg 922extending below the upper edge of the end walls of the container 321when the divided element 513a and the container 321 are interengaged.The container 321 is provided with a transverse projection 923 whichbecomes engaged with leg 922 when the divided element 513a is engagedwith the container. The leg 922 is deflected laterally when the element513a is inserted into the container and it undergoes a snap-type actionfor engaging the projection 923 after the divided element 513a has beenfully engaged within the container 321. Sealing strips 930 are mountedat the bottom edge of the divided element 513a for being simultaneouslycompressed against the shelf 932 on the container 321 and the sealingaction is maintained by the pressure exerted by the engagement of leg922 with the projection 923 after the divided element 513a has beenfully inserted into the container 321.

Although the particular locking and sealing arrangement has been shownin conjunction with the divided element having hollow inner members 902,it is to be understood that the divided element may be of the form andconstruction as shown in any of the previous embodiments.

FIG. 38 shows an arrangement wherein the reactivity of the agent andactive substance can be determined by measuring a reaction zone.Specifically, in FIG. 38 the divided element 1003 is essentiallyconstituted by a plurality of juxtaposed elements of the type as shownin FIG. 10. In this embodiment each of the divided sections 1004 formedin the divided element 1003 is provided with a lining 1014 along onlyone of the narrow end walls. When the divided element 1003 is introducedinto the container so that the active substance in the lining 1014 onthe end wall can diffuse into the substrate, the reaction takes placewithin each of the sections 1004 confined between relatively long walls1020. These sections are relatively long and narrow rectangles and thediffusion of the active substance is guided by the shape of the sections1004 along the longitudinal axis of the rectangular section. The lengthof the reaction zone is indicative of the degree of reaction between theactive substance and the agent. Instead of the provision of lining 1014on the narrow end wall, the active substance, for example, a liquid canbe added by using a perforated lamella as shown by way of example inFIGS. 4 and 9, or an article containing active substance may be placedon or in the substrate close to the narrow end wall which in this caseis devoid of active substance. The article may be a disc. The oppositenarrow end walls of the divided element 1003 may be omitted.

FIG. 39 shows a dispenser 47, for example, of paper discs 51 or tabletsor other suitable bodies containing substances to be added to thesubstrate, in combination with the divided element 13a and container 21of FIGS. 16 and 17. The discs 51 can be used to supply active substancein conjunction with the supply of active substance from the linings onthe walls of the divided element 13a or in replacement of the supply ofactive substance from the linings on the divided element. In the lattercase, the linings may be retained but will be bare or the linings can beomitted. Also, instead of linings, when used, on the walls of thedivided element, a distributing means of the type as shown in FIGS. 4and 9 can be employed. Additionally, the dispenser 47 can be employedwith the embodiment shown in FIGS. 35-37 in which case the discs 51 willbe deposited into the hollow bodies 902 and supported therein. A base 57is shown for centering and supporting container 21 containing substrate22. The dispenser 47 is supported by means of an arm 48 on an upright 49on the base 57 for pivotal movement in a horizontal plane from theillustrated active position, which is determined by a stop, to aninactive position where the container can be inserted and removed. Thedispenser 47 contains vertical dispensing tubes 50 adapted to holdstacks of discs 51. The stacks of discs normally rest on a base plate52, which is fitted with outlets 53 laterally displaced in relation tothe relevant dispensing tube 50. The bottom disc in each stack is movedto the associated aperture by moving a pusher 55 against the bias of areturn spring 56. The pusher 55 is provided with a straighttranslational guiding support.

The base 57 and the dispenser 47 can be aligned with one another suchthat the outlets 53 in the operating positions are directly above theopen upper ends of the sections 23 of the divided element 13a. Each setof discs released will fall parallel from the outlets 53 and bepositioned on the substrate 22 as shown at 51' in FIG. 39.

It is also possible within the scope of the invention to employ amagazine containing the supporting elements in a dispenser similar todispenser 47 and to provide means for mechanically and/or magneticallypressing the supporting elements into the container with the substrate.It is to be understood that the supporting elements and the conformingcontainers may be made relatively small in size so that a miniaturizedsystem may be provided, although any appropriate size may be used.

Although, FIG. 39 has been described in conjunction with deposit of thediscs onto the substrate after the assembly of the divided element andthe container, it is possible in accordance with the invention to addthe discs to the divided element into the hollow bodies 900 of thedivided element 413a in FIGS. 35-37 or into cavity 809 of FIG. 34 beforethe divided element is inserted into the container, although suchadditions by means of a dispenser should preferably be made afterassembly.

The container can be provided with a flap or other suitable attachment(not shown) on which identification and information data can be providedin accordance, for example, with the sample and person from which theagent added to the substrate is derived and with the test involved. Theprinciples disclosed in U.S. Pat. No. 3,975,716 may be used for dataidentification and information in this respect.

The disclosed embodiments merely serve to illustrate the invention andnot to limit it, inasmuch as variations and modifications will becomeapparent which will fall within the scope of the invention as defined inthe appended claims.

What is claimed is:
 1. Apparatus for carrying out biological andchemical diffusion tests in microbiological, serological, immunological,and clinical-chemical work, in which at least one active substance iscontacted with a substrate containing an agent, the reactivity of theagent and active substance being the subject of investigation, saidapparatus comprising a container containing the substrate, a dividedcarrying body with respective separated sections in sealed contactwithin the container such that the sections form separate regions withisolated substrate portions in each of said regions, said carrying bodypenetrating into said substrate in each of said regions and meanssupported by said carrying body for introducing said active substanceinto respective isolated substrate portions where the carrying bodypenetrates into the substrate by discharging said active substance intothe penetrated substrate portions by diffusion whereby the action insaid substrate is restricted to said separate regions.
 2. Apparatus asclaimed in claim 1 wherein said carrying body has lower edges and is insealed contact with the container at said lower edges.
 3. Apparatus asclaimed in claim 2 comprising sealing means for forming a liquid-tightseal between the lower edges of the carrying body and said container. 4.Apparatus as claimed in claim 1 wherein said means for introducingactive substance into the isolated substrate portions comprises an innerbody supported within each section of the carrier body.
 5. Apparatus asclaimed in claim 4 wherein each inner body is spaced from the walls ofthe respective section of the carrier body.
 6. Apparatus as claimed inclaim 5 wherein each said inner body projects axially within itsrespective section.
 7. Apparatus as claimed in claim 4 wherein saidinner body has an outer surface facing said walls of the respectivesection of the carrier body and the active substance is diffused fromthe inner body into the substrate from said outer surface towards saidwalls.
 8. Apparatus as claimed in claim 1 wherein said active substancecomprises a substance for modifying growth of said agent.
 9. Apparatusas claimed in claim 1 wherein said means for introducing activesubstance into the isolated substrate portions comprises a perforatedinner part.
 10. Apparatus as claimed in claim 9 wherein said inner partis spaced from the wall of the respective separated section anddischarge of the active substance is effected from the space between thewall and the inner part to said substrate via the perforated inner part.11. Apparatus as claimed in claim 1 comprising means for photometricallyanalyzing each of said separate regions to determine the extent of thereactivity between the agent and the active substance therein. 12.Apparatus as claimed in claim 1 wherein said active substance iscontained in a dispensible article, said apparatus further comprisingdispenser means for dispensing the dispensible article into the meanswhich is supported by the carrying body for introducing the activesubstance into the isolated substrate portions.
 13. The invention asclaimed in claim 12 wherein said active substance is contained in adispensible article, the combination further comprising dispenser meansfor dispensing the dispensible article into the means which is supportedby the carrying body for introducing the active substance into theisolated substrate regions.
 14. For use in combination with a substratecontaining an agent to carry out biological and chemical diffusion testsin microbiological, serological, immunological, clinical-chemical andsimilar laboratory work: a carrying body including separating partitionsdefining separate sections in said body, said carrying body havingopenings which lead into said separate sections, a container for saidsubstrate, said carrying body and container being relatively engageableto cause said substrate to be introduced into said openings and formisolated regions of said substrate in said sections in which saidcarrying body is immersed to a given depth in the substrate, and meanssupported by said carrying body for diffusing an active substance intorespective isolated regions in the vicinity of immersion of the body inthe substrate for producing a substantial equilibrium concentration ofthe active substance in said confined area, the reactivity of the activesubstance and agent being the subject of the invention.
 15. Theinvention as claimed in claim 14 wherein said means for diffusing activesubstance comprises an inner body supported within each section of thecarrier body.
 16. The invention as claimed in claim 15 wherein eachinner body is spaced from the walls of the respective section of thecarrier body.
 17. The invention as claimed in claim 16 wherein saidinner body has an outer surface facing said walls of the respectivesection of the carrier body and the active substance is diffused fromthe inner body into the substrate from said outer surface towards saidwalls.
 18. The invention as claimed in claim 14 comprising means forphotometrically analyzing each of said isolated regions to determine theextent of the reactivity between the agent and the active substancetherein.
 19. The invention as claimed in claim 14 wherein the means bywhich the substrate is introduced into said openings and forms isolatedregions of said substrate on said sections comprises an outer containercontaining said substrate and in which outer container said carryingbody is engaged in sealed relation.
 20. The invention as claimed inclaim 19 wherein said inner body is perforated and discharge of activesubstance is effected to the substrate via the perforated inner body.21. The invention as claimed in claim 14 wherein said active substancecomprises a substance for modifying growth of said agent.
 22. Apparatusfor carrying out biological and chemical diffusion tests inmicrobiological, serological, immunological, and clinical-chemical work,in which at least one active substance is contacted with a substratecontaining an agent, the reactivity of the agent and active substancebeing the subject of investigation, said apparatus comprising acontainer containing the substrate, an elongated carrying body includingat least one first wall and two second walls, the second walls beingrelatively long compared to said first wall, said carrying body being insealed contact within the container such that the body forms a regionwith isolated substrate portion, and means on said first wall forintroducing said active substance into the isolated substrate portion bydiffusion whereby the diffusion is guided by the relatively long secondwalls and the length of a reaction zone measured from the first wall isindicative of the reactivity of the agent and active substance. 23.Apparatus as claimed in claim 22 wherein said carrying body has loweredges and is in sealed contact with the container at said lower edges.24. Apparatus as claimed in claim 22 wherein a plurality of elongatedcarrying bodies are secured together in juxtaposed relation.
 25. Amethod of carrying out biological and chemical diffusion tests inmicrobiological, serological, immunological, and clinical-chemical work,in which at least one active substance is contacted with a substratecontaining an agent in a container, the reactivity of the agent andactive substance being the subject of investigation, said methodcomprising forming a divided carrying body with respective separatedsections, sealably contacting said carrying body with the container suchthat the sections form separate regions with isolated substrate portionsin each of said regions, the carrying body being immersed in thesubstrate and introducing said active substance into the isolatedsubstrate portions with said carrying body maintained in sealed contactwith the container, said introducing of said active substance into theisolated substrate portions being effected by discharging said activesubstance from within said carrying body into the substrate portions bydiffusion in the region of immersion of the carrying body in thesubstrate whereby the action in said substrate is restricted to saidseparate regions.
 26. A method as claimed in claim 25 wherein thecarrying body is in sealed contact with the container at the lower edgeof the carrying body.
 27. A method as claimed in claim 26 comprisingforming an inner body within each section of the carrying body immersedin the respective isolated substrate portions for the introduction ofthe active substance into the isolated substrate portions.
 28. A methodas claimed in claim 25 wherein said active substance is dispensed in adisc into said carrying body.
 29. A method as claimed in claim 25comprising effecting at least one secondary application of at least onedifferent active substance to the substrate subsequent to the time afterthe first substance and agent have been brought into contact with oneanother.
 30. A method as claimed in claim 29 wherein the substanceapplied to the surface of the substrate is in particulate form.
 31. Amethod as claimed in claim 30 wherein the substance is a bacterium. 32.A method as claimed in claim 29 wherein the substance applied in thesecondary application is red blood cells.
 33. A method as claimed inclaim 29 wherein the substance applied in the secondary application iscoated onto inert particles.
 34. A method as claimed in claim 25comprising photometrically analyzing each of said regions to determinethe extent of the reactivity between the agent and the active substance.35. A method as claimed in claim 34 comprising introducing a distinctionpromoting substance to said substrate to enhance the photometricanalyzation of the regions.
 36. A method as claimed in claim 34comprising agitating the substrate after introduction of the activesubstance thereinto and prior to photometric analysis.
 37. A method asclaimed in claim 34 comprising incubating the substrate to promote thereactivity of the agent and active substance.
 38. A method as claimed inclaim 34 wherein the photometric analysis is effected by lightscattering.
 39. A method for carrying out immunological and similarbiochemical examinations in which at least one active substance iscontacted with an agent in a container containing a solid or semi-solidsubstrate, the active substance being selected from the group consistingof the class of antigens and the class of antibodies, said agent being atest dose of a substitutent from the other of said classes, said methodcomprising forming a divided carrying body with respective separatedsections, immersing said carrying body into the substrate in thecontainer such that said sections form separate regions with isolatedsubstrate portions in each of said regions, introducing said activesubstance into the isolated substrate portions with said carrying bodymaintained in the container, said introducing of said active substanceinto the isolated substrate portions being effected by discharging saidactive substance from within said carrying body into the substrateportions by diffusion in the region of immersion of the carrying body inthe substrate and adding the agent to the substrate at the surfacethereof after the carrying body is introduced into the container suchthat the antigens and antibodies undergo agglutination during reactionand form aggregates at the surface of the substrate wherebyimmunological examination of said classes can be effected at the surfaceof the substrate.
 40. Apparatus for carrying out biological and chemicaldiffusion tests in microbiological, serological, immunological andclinical chemical work comprisingsupport means, a microporous substrateon said support means and including a quantity of agent in relation tothe amount of substrate, said support means confining said substrate toform a confined reaction zone therein, and distributing means fordistributing active substance in liquid form into said substrate, saiddistributing means including a layer of porous material also confined bysaid support means for uniformly distributing the active substance intothe substrate to provide a determined uniform quantity of activesubstance in the confined amount of substrate, said support means havingan opening through which the active substance can be added to saidporous material and thereby to the confined substrate, said quantity ofagent and distributing means cooperatively forming a system in which anamount of active substance can be related with a given volumetric amountof substrate to provide rapid diffusion equilibrium in said confinedsubstrate, said layer of porous material of said distributing meanshaving a surface contact area with said substrate which is coextensivetherewith and through which the quantity of active substance isuniformly distributed into said substrate.
 41. For use in combinationwith a substrate containing an agent to carry out biological andchemical diffusion tests in microbiological, serological, immunological,clinical-chemical and similar laboratory work: a tubular carrying bodyintroducible into the substrate, and means associated with said body fordiffusing an active substance from said body into the substrate when thetubular body is introduced therein, the reactivity of the activesubstance and agent being the subject of the investigation, said tubularbody comprising magnetically responsive material for the application ofmagnetic force thereto to move the body in the substrate.
 42. Theinvention as claimed in claim 41 wherein said tubular body comprises awall, said magnetically responsive material being in said wall.
 43. Theinvention as claimed in claim 41 wherein said tubular body is vacuumpacked prior to its introduction into the substrate.
 44. The inventionas claimed in claim 41 wherein said tubular body has opposite open ends.45. The invention as claimed in claim 41 wherein said active substanceis supported within said tubular body.
 46. For use in carrying outbiological and chemical diffusion tests in microbiological, serological,immunological, clinical-chemical and similar laboratory work: a carryingbody including a wall which is shaped to confine a substantially closedarea on one side thereof, means comprising an active substance held onsaid wall in a position facing into said closed area for diffusing saidactive substance from said body into the closed area, and a substratecontaining an agent confined within said closed area and in which saidmeans is immersed such that said active substance is diffused into thesubstrate along the depth of immersion therein for producing asubstantial equilibrium concentration of the active substance in saidconfined area, the diffusion of active substance into the substratetaking place from said wall radially inwards along a diffusion frontparallel to said wall towards an axis of the body, the diffusion beingeffected over the depth of penetration of the body into the substrate asmeasured along said axis, the reactivity of the active substance andagent being the subject of the investigation.
 47. The apparatus asclaimed in claim 46 wherein said carrying body includes separatingpartitions therein to form a plurality of separated closed areas, thelinings active substances in the respective closed areas havingsuccessively varying concentrations.
 48. Apparatus for carrying outbiological and chemical diffusion tests in microbiological, serological,immunological, clinical-chemical and similar laboratory work comprisinga carrying body including a wall which is shaped to confine asubstantially closed area on one side thereof, means comprising a liningof an active substance on said wall facing into said closed area fordiffusing an active substance from said body into said area to carry outin said closed area a biological or chemical diffusion reaction, andmeans for introducing into said closed area an agent related to thereaction effected therein, said agent extending to a given depth on saidcarrying body, said lining extending a height along said wall to diffusesaid active substance into said agent along the entire depth of theagent in said carrying body, the diffusion of active substance into thesubstrate taking place from said lining along a diffusion front parallelto said wall along the depth of the agent in the carrying body.
 49. Theinvention as claimed in claim 47 wherein said means extends a sufficientheight along said wall to diffuse said active substance into said agentalong the entire depth of the agent in said carrying body.
 50. Theinvention as claimed in claim 49 wherein said lining is a coating onsaid wall.
 51. For carrying out biological and chemical diffusion testsin microbiological, serological, immunological, clinical-chemical andsimilar laboratory work: a hollow carrying body shaped to confine asubstantially closed area on one side thereof, a substrate containing anagent in said carrying body, and means penetrating into said substratefor diffusing an active substance into the substrate for producing asubstantial equilibrium concentration of the active substance in saidconfined area, said closed area having an exposed surface, said meanspenetrating into said substrate to form a space in said substratebetween said means and said hollow carrying body which space surroundssaid means, the reactivity of the substance and agent being the subjectof the investigation.
 52. The invention as claimed in claim 51 whereinsaid means extends axially within said carrying body.
 53. The inventionas claimed in claim 52 wherein said means comprises a central body withmeans for releasing active substance at the outer surface thereof. 54.The invention as claimed in claim 51 wherein said substrate is a liquid.55. The invention as claimed in claim 54 wherein said means is immersedin said liquid.
 56. The invention as claimed in claim 51 wherein saidmeans comprises a solid body.
 57. The invention as claimed in claim 56wherein said active substance is a coating on said solid body.
 58. Theinvention as claimed in claim 51 wherein said means comprises a hollow,perforated body.
 59. The invention as claimed in claim 51 wherein saidhollow body comprises a wall and said means comprises a hollowperforated inner element spaced from said wall and defining said spacetherebetween, the active substance being introducible into said spacefor diffusion into the substrate via the perforated inner element. 60.For use in carrying out biological and chemical diffusion tests: acarrying body having an outer surface, means containing an activesubstance covering at least a portion of said outer surface of saidcarrying body, means including a medium containing an agent, thereactivity of the active substance and agent being the subject ofinvestigation, said carrying body being introducible into said medium todefine a confined region of said medium with which the active substanceis in contact for producing substantially rapid equilibriumconcentration of the active substance in said confined region and acontainer for said medium, said carrying body being introduced into saidcontainer to penetrate into the medium and form said confined region ofsaid medium between the carrying body and said container.
 61. Thecombination as claimed in claim 60 comprising magnetically responsivemeans incorporated in said carrying body and means for applying magneticforce to said magnetically responsive means to draw said carrying bodythrough said substrate.
 62. The combination comprising: an article forcarrying out testing, said article comprising a test dose of an activesubstance which may react with an agent of a medium when the article isbrought into contact with the medium, and magnetically responsive meansincorporated with said test dose as a unitary body; and means forapplying a magnetic force to said magnetically responsive means.
 63. Amethod for testing reactions comprising forming a substrate ofprescribed composition and including an agent, forming an articlecontaining an active constituent whose reactivity with the agent of thesubstrate is to be determined by the production of a detectable resule,incorporating magnetically responsive material in said article, bringingthe article and substrate into contact with one another and applying anexternal magnetic force to said article.
 64. A method as claimed inclaim 63 wherein said magnetic force acts to draw the article throughthe substrate.
 65. Apparatus for carrying out biological and chemicaldiffusion tests in microbiological, serological, immunological,clinical-chemical and similar laboratory work: comprising a substratecontaining an agent, and means forming an isolated reaction region forsaid substrate and including a coated carrying body introducible intosaid agent in said isolated region for diffusing active substance fromthe coating on the carrying body into the substrate, said carrying bodybeing introducible into said substrate as a free body to a given depth,the active substance being diffused into the substrate over the depth ofintroduction of the carrying body into the substrate for producing asubstantial equilibrium concentration of the active substance in saidagent, the reactivity of the active substance and agent being thesubject of the investigation.